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Abstract

Background—Mutations in the gene G4.5 result in a wide spectrum of severe infantile cardiomyopathic phenotypes, including isolated left ventricular noncompaction (LVNC), as well as Barth syndrome (BTHS) with dilated cardiomyopathy (DCM). The purpose of this study was to investigate patients with LVNC or BTHS for mutations in G4.5 or other novel genes.

Methods and Results—DNA was isolated from 2 families and 3 individuals with isolated LVNC or LVNC with congenital heart disease (CHD), as well as 4 families with BTHS associated with LVNC or DCM, and screened for mutations by single-strand DNA conformation polymorphism analysis and DNA sequencing. In 1 family with LVNC and CHD, a C→T mutation was identified at nucleotide 362 of α-dystrobrevin, changing a proline to leucine (P121L). Mutations in G4.5 were identified in 2 families with isolated LVNC: a missense mutation in exon 4 (C118R) in 1 and a splice donor mutation (IVS10+2T→A) in intron 10 in the other. In a family with cardiomyopathies ranging from BTHS or fatal infantile cardiomyopathy to asymptomatic DCM, a splice acceptor mutation in exon 2 of G4.5 (398-2 A→G) was identified, and a 1-bp deletion in exon 2 of G4.5, resulting in a stop codon after amino acid 41, was identified in a sporadic case of BTHS.

Conclusions—These data demonstrate genetic heterogeneity in LVNC, with mutation of a novel gene, α-dystrobrevin, identified in LVNC associated with CHD. In addition, these results confirm that mutations in G4.5 result in a wide phenotypic spectrum of cardiomyopathies.

Left ventricular noncompaction (LVNC) is reportedly rare and has been thought to be due to an arrest of myocardial morphogenesis.1 This disorder is characterized by a hypertrophic LV with deep trabeculations and with poor systolic function, with or without associated LV dilation.23 In some cases, the right ventricle is also affected. No congenital heart defects (CHDs) are seen in these patients. Similar myocardial patterns are occasionally reported postnatally in association with congenital heart anomalies2 such as ventricular septal defects (VSDs), pulmonic stenosis, and atrial septal defects. This has been called nonisolated LVNC or LVNC associated with CHD. Although the genetic cause of LVNC with CHD is unknown, recent studies using genetic linkage and mutation analysis identified mutations in the gene G4.5 in patients with isolated LVNC.4G4.5 was initially associated with Barth syndrome (BTHS),5 a disease first described by Neustein et al6 and Barth et al,7 and encodes a novel protein family called the tafazzins. BTHS is a clinical association that includes skeletal myopathy and either endocardial fibroelastosis or dilated cardiomyopathy (DCM), neutropenia, abnormal mitochondria, growth retardation, abnormal cholesterol metabolism, lactic acidosis, elevated urinary 3-methylglutaconic acid and 2-ethylhydracrylic acid, and X-linked recessive inheritance. Mutations in G4.5 result in not only BTHS but also other X-linked infantile cardiomyopathies, including LVNC,4 X-linked infantile cardiomyopathy,89 and X-linked endocardial fibroelastosis.8

Myocardial disorders affecting systolic function but without deep trabeculations have also been seen in animal models or human patients with mutations disrupting the dystrophin gene1011 or its interaction with other proteins, such as the dystrophin-associated glycoprotein complex (DAPC)12 or the sarcomere (actin, muscle LIM protein).1314 In this report, we identify a mutation of the gene for α-dystrobrevin, a cytoskeletal protein in the DAPC, in a family with LVNC with CHD. In addition, we describe novel G4.5 mutations in individuals with isolated LVNC, as well as in patients with BTHS and other forms of DCM, including a family with widely variant phenotypic presentations, thus extending the phenotypic spectrum of patients with mutations in G4.5.

Methods

Clinical Diagnostic Criteria

LVNC was diagnosed by echocardiographic criteria, including (1) LV hypertrophy with deep endomyocardial trabeculations in ≥1 ventricular wall segments, (2) reduced LV systolic function, and (3) presence or absence of LV dilation. Cardiac structure was evaluated, and cardiac anomalies that exhibit a similar myocardial pattern of persistent sinusoids, such as pulmonary atresia, were excluded. After a proband was identified, a family history was obtained, and all potentially informative family members underwent physical examination, chest radiograph, ECG, and echocardiogram.

Stringent clinical criteria were used for diagnosis of BTHS in the probands. In all cases, the probands were required to have the following features: male infants, sporadic or X-linked inheritance, DCM or dilated hypertrophic cardiomyopathy, neutropenia, and 3-methylglutaconic aciduria. The clinical evaluation of each proband included physical examination and echocardiography (2D, M-mode, color Doppler) with evaluation of cardiac structure and with standard measurements of LV size, function (shortening fraction, ejection fraction), and valvular regurgitation. In addition, chest radiography, electrocardiography, urine organic acids, and complete blood count with differential were performed. Family members were screened by physical examination, chest radiograph, ECG, echocardiogram, urine organic acid analysis, and complete blood count with differential.

After informed consent, blood was obtained for the development of lymphoblastoid cell lines15 and DNA extraction by use of QIAamp DNA extraction kits. Age-, ethnicity-, and sex-matched negative control patients were recruited, and blood was obtained for DNA extraction after informed consent.

Single-Strand Conformational Polymorphism Analysis

Mutation analysis was performed as previously described.15 Primers were designed to amplify the genes encoding G4.5 (Table 1⇓) and α-dystrobrevin (Table 2⇓) in an exon-by-exon manner. Radioactive PCR was performed as previously described.15 In brief, after a 5-minute denaturation step at 94°C, 35 cycles of amplification (94°C for 30 seconds, X°C for 30 seconds, and 72°C for 20 seconds, where X represents the annealing temperature shown in the respective Table⇓) were performed. This was followed by a 72°C incubation for 3 minutes. After PCR amplification, the samples were denatured and analyzed by polyacrylamide gel electrophoresis and autoradiography.15

Primers, PCR Condition, and PCR Products for the Amplification of Each Exon of α-dystobrevin

Sequence Analysis

Normal and aberrant single-strand conformational polymorphism (SSCP) conformers from each exon were excised directly from dried gels, purified, and sequenced according to the ABI Big Dye Terminator Cycle Sequencing protocol and either an ABI 373 or ABI 310 Automated Sequencer.15

BLAST search was used to identify homology between the sequences obtained from patients and to evaluate sequence conservation across species. Protein structural prediction was performed by the Garnier-Osguthorpe-Robson plot method.16

Results

Patient Characteristics and Mutation Screening

LVNC With CHD

Family NLVNC-09 (Figure 1A⇓): A 4-generation Japanese family with 6 affected individuals, including 5 with LVNC associated with CHD and 1 with isolated LVNC (proband III-2), was identified. The patients’ ages ranged from 2 days to 69 years. All patients with CHD had ≥1 VSDs. In 1 child (IV-2), a patent ductus arteriosus was noted, and another child (IV-3) was identified with a hypoplastic LV. One child (IV-1) died with a hypoplastic left heart and hydrops fetalis diagnosed 2 days after birth.

Pedigrees of families reported in this study. Solid circles and squares indicate affected females and males, respectively; open circles and squares, unaffected; circles with central dots, female carriers; and slashes, death. Probands are indicated by arrows.

Although the pattern of inheritance was determined to be autosomal dominant or mitochondrial, an X-linked pattern could not be completely excluded. For that reason, genetic screening for G4.5 mutations was undertaken. No mutations were found by either SSCP analysis or direct sequencing.

Mutation analysis of α-dystrobrevin identified an abnormal SSCP conformer in affected members of NLVNC-9 (Figure 2A⇓). Manual (Figure 2B⇓) and automated (Figure 2C⇓) sequencing identified a C→T substitution at position 362 of exon 3, resulting in an amino acid change from proline to leucine at codon 121 (P121L, Table 3⇓). The unaffected members of this family did not carry this mutation. Three hundred age- and sex-matched controls (200 Japanese, 100 white) were screened as well, and no mutations were identified.

Detection of mutations in α-dystrobrevin. SSCP analysis (A) of NLVNC-09 identifies an abnormal band (arrow) in affected members of this family (see pedigree above each patient lane). Automated sequencing (B) identifies a point mutation (C→T) at nucleotide 362 within exon 3 of α-dystrobrevin in affected individuals compared with wild-type normal sequence.

Comparison of human and mouse α-dystrobrevin demonstrates this nucleotide (362) and codon (121) to be conserved: there is >90% homology in this region of the dystrobrevin gene. Protein sequence analysis predicted that the amino acid substitution will result in the reduction of an α-helix by 2 amino acids and the removal of a loop in this portion of the protein, which encodes the calcium-binding EF-hand domain, possibly resulting in a significant secondary structural change.

Family NLVNC-10: The second family with LVNC with CHD (Figure 1B⇑, NLVNC-10) was also identified in Japan. This 2-generation family includes 3 affected individuals; in 2 patients (including the proband I-2), an associated atrial septal defect was identified (I-2 and II-2), and the other patient had a small VSD that closed spontaneously (II-3). All affected patients were female, and all are alive. The pattern of inheritance is autosomal dominant or mitochondrial. No mutations in α-dystrobrevin or G4.5 were found in NLVNC-10, suggesting genetic heterogeneity in LVNC associated with CHD.

Sporadic cases: In the 3 sporadic cases from the United States, all children presented with LVNC early in life (1 week, 3 weeks, 2 months); 2 were female and 1 male. In all 3 patients, a VSD was identified. The male child also had pulmonic stenosis. All are still alive. No mutations in α-dystrobrevin or G4.5 were found.

Isolated LVNC

Family BSG (Figure 1C⇑): This family consisted of a 33-year-old mother and 26-year-old father; there were no children other than the 5-month-old male proband with isolated LVNC associated with a dilated, mildly hypertrophic heart (Figure 3⇓), with poor systolic function on echocardiogram and clinical heart failure. Neutropenia and 3-methylglutaconic aciduria were also identified. Maternal family history was notable for 1 sibling (healthy sister) and no family history of heart disease or hematologic disease. The mother’s echocardiogram was normal.

Family BSL (Figure 1D⇑): This Vietnamese family included a 23-year-old mother and 26-year-old father. The proband was the only child born to this couple, and he presented in heart failure in infancy. An echocardiogram demonstrated a severely dilated LV with deep trabeculations consistent with isolated LVNC, and systolic function was poor; neutropenia and 3-methylglutaconic aciduria were also identified. The child’s mother was adopted, and therefore no family history was available. Her health was good, with no evidence of heart disease, and her echocardiogram was normal.

SSCP analysis using primers for exon 10 of G4.5 identified an abnormal conformer in the proband and his mother. DNA sequence analysis identified a T→A substitution at the intron 10 splice donor site (IVS10+2T→A, Table 3⇑).

Dilated Cardiomyopathy with BTHS

Family BSH (Figure 1E⇑): This infant boy (3 months) presented with signs and symptoms of congestive heart failure and low cardiac output. The echocardiogram demonstrated hypertrophic dilated cardiomyopathy with severely reduced LV function. Neutropenia and 3-methylglutaconic aciduria were also identified. Evaluation of family members identified 2 other male children, previously diagnosed with pure DCM by echocardiography in infancy, who had died suddenly with DCM and heart failure. The parents were in good health, and the mother’s family history was negative for heart disease or sudden death. Her echocardiogram was normal. She had 2 sisters who were clinically well.

Using primers for exon 2 of G4.5, SSCP analysis identified an abnormal conformer in affected and carrier individuals. Sequence analysis demonstrated a deletion of cytosine (TCCA→TCA) at nucleotide 123 (codon 41), resulting in a frameshift that changed codon 42 from a leucine to a premature stop codon, thereby truncating the protein product (Table 3⇑).

Family BSD (Figure 1F⇑): The proband was a 15-year-old diagnosed with signs and symptoms of congestive heart failure and low cardiac output. Echocardiograms demonstrated LV dilation and severely reduced LV function. The members of this family were screened by echocardiography, which allowed identification of multiple affected males, including 2 infants (Figure 1F⇑, III-3, III-4). For this reason, all patients were screened for organic aciduria and metabolic derangements. These studies identified lactic acidosis in 2 children (Figure 1F⇑, III-2, III-7), cyclic neutropenia in 3 individuals (Figure 1F⇑, III-3, III-4, III-7), and 3-methylglutaconic aciduria in 2 children (Figure 1F⇑, III-4, III-7). In 2 children, protein-C deficiency was diagnosed (Figure 1F⇑, III-3, III-4). The members of this family had diagnoses ranging from classic BTHS to fatal infantile DCM to late-onset symptomatic and asymptomatic DCM.

An abnormal conformer in exon 2 of G4.5 was also detected in this proband (Figure 4A⇓). DNA sequence analysis demonstrated an A→G substitution at the splice acceptor site (398-2 A→G) of exon 2 (Figure 4B⇓ and 4C⇓). Subsequently, SSCP and sequence analysis of extended family members demonstrated the identical mutation in males with DCM (with or without metabolic abnormalities, Table 3⇑) and female carriers.

Detection of mutations in G4.5. SSCP analysis of family BSD (A) identifies an abnormal conformer (arrow) in affected males and carrier females, as seen in pedigree. Manual (B) and automated (C) DNA sequence identifies an A→G substitution at splice acceptor site of exon 2 (nucleotide 398) of G4.5.

None of these G4.5 mutations were identified in 200 control patient samples.

Discussion

Over the past few years, our understanding of the molecular basis of DCM has increased considerably, with the description of mutations in several of the structural proteins, including dystrophin,17 lamin A/C,18 desmin,19 cardiac actin,14 and δ-sarcoglycan.20 In addition, mutations have been described in the gene G4.5 in patients with classic BTHS,521 as well as in patients with infantile DCM8 and isolated LVNC.4 Here, we report the identification of novel mutations in G4.5 in patients with isolated LVNC. In patients with LVNC associated with CHD, however, no mutations were identified in G4.5. Instead, we identified a mutation in the calcium-binding EF-hand domain of α-dystrobrevin in 1 family.

The α-dystrobrevin gene is alternatively spliced, resulting in multiple isoforms of dystrobrevin (α, β, γ), with different tissue distributions,22 of which only α-dystrobrevin is expressed in the heart. α-Dystrobrevin is a member of the DAPC, which is composed of 3 subcomplexes: the dystroglycan complex, the sarcoglycan complex, and the cytoplasmic complex, which includes the syntrophins and dystrobrevins.23 The DAPC, which is located at the sarcolemma, connects the cysteine-rich and C-terminal domains of dystrophin with β-dystroglycan and the cytoplasmic complex, respectively. β-Dystroglycan is a transmembrane protein that binds to the laminin-binding protein α-dystroglycan in the extracellular matrix.24 At the N-terminus, dystrophin binds to actin. These interactions effectively link the extracellular matrix to the dystrophin-based cytoskeleton of the muscle fiber at the C-terminus and to the contractile apparatus at the N-terminus. Furthermore, α-dystrobrevin links the DAPC to the signaling protein neuronal nitric oxide synthase (nNOS).25 Disruption of these links results in severe muscle wasting or cardiac muscle pathology.1126 For example, dystrophin mutations cause Duchenne muscular dystrophy27 or X-linked dilated cardiomyopathy,17 whereas mutations in actin have been shown to cause either DCM or hypertrophic cardiomyopathy,1428 and mutations in δ-sarcoglycan result in limb-girdle muscular dystrophy29 or DCM.20 Mutations in α-dystrobrevin have been shown to result in muscular dystrophy in humans30 and have also been found to cause skeletal myopathy and cardiomyopathy in mice.31 Of particular interest is the description by Grady and colleagues32 of a mouse deficient in α-dystrobrevin, derived by homozygous deletion of exon 3, where reduced signaling via nNOS results in skeletal and cardiac (degenerating myocytes, mononuclear cell infiltration, and fibrosis) myopathies. These data suggest that the region of the gene encoded by exon 3, the same region as mutated in the patient described here, is essential for functionality of the protein. We are in the process of generating transgenic mice to confirm that the mutation identified in this patient is disease-causing.

The α-dystrobrevin mutation described here results in a phenotype of dilated hypertrophic cardiomyopathy with deep trabeculations, associated with congenital heart disease, consistent with the criteria for LVNC. There is considerable variability in disease development in this family, however, with congenital disease ranging in severity from a simple VSD to hypoplastic left heart syndrome; in 1 patient, isolated LVNC without CHD was also notable. Several mechanisms could account for these observations, including environmental factors and acquired traits, as well as the existence of modifier genes and polymorphisms that could modulate phenotypic expression.3334 For example, for the ACE insertion/deletion (ACE I/D) polymorphism, the D allele in patients with hypertrophic cardiomyopathy is associated with a high incidence of sudden death.33 It is also likely that the various forms of CHD occur because of alterations in the signaling pathways that α-dystrobrevin participates in, including the interactions with the NOS and transforming growth factor-β pathways. It has been well described previously that transcription factor abnormalities can cause CHD,35 and the forms of CHD may be quite variable, as occurs with heterotaxy syndrome, with wide differences in severity. Although it is unclear at present what specific mechanism is involved, we would suggest that perturbations in these pathways lead to developmental errors that are dependent on genetic background and developmental timing. Because myocardial development occurs very early in human development, a variety of interactions later in development are likely to modify the results of the initial insult.

The novel mutations identified in G4.5 in patients with LVNC or BTHS and in patients with relatively late-onset DCM included nonsense and missense mutations. Several important points are now becoming apparent with regard to G4.5 mutations. First, many affected individuals develop severe infantile disease and succumb. The gene defect usually differs among families, however, and thus far there do not appear to be obvious genotype:phenotype correlations that allow the differentiation of clinical course to be predicted. Second, the cardiac phenotypes that occur as a result of G4.5 mutations may vary significantly. The cardiac manifestations include DCM, endocardial fibroelastosis, LVNC, and dilated hypertrophic cardiomyopathy. In addition, this phenotype can differ among family members and change over time, possibly in response to therapy. Finally, the systemic manifestations of BTHS are equally unpredictable. In some children, sudden death occurs. It is likely that modifier genes are involved in determining the phenotype and clinical severity.

We suggest that studies of patients with myocardial disorders having prominent systolic dysfunction should include evaluation of members of the cytoskeleton-sarcolemma complex, as well as G4.5, as candidate genes, and when associated with CHD, signaling pathways/transcription factors should be targeted as well.

Acknowledgments

This work was supported by grants from the Abercrombie Cardiology Fund of Texas Children’s Hospital, the Texas Children’s Hospital Foundation Chair in Pediatric Cardiovascular Research, the American Heart Association (Texas Affiliate and National Center), the Howard Hughes Medical Institute, the National Institutes of Health, National Heart, Lung, and Blood Institute, and the Japanese Ministry of Education, Science, and Culture. Fukiko Ichida, MD, is supported by grants from the Ministry of Education, Science, and Culture in Japan. Neil E. Bowles, PhD, is supported by a grant from the American Heart Association, Texas Affiliate, and by the Abercrombie Cardiology Fund of Texas Children’s Hospital. Karla R. Bowles, PhD, is a Howard Hughes Medical Institute Predoctoral Fellow. William J. Dreyer, MD, is supported by grants from the American Heart Association, National Center, and by the Abercrombie Cardiology Fund of Texas Children’s Hospital. Jeffrey A. Towbin, MD, is supported by the Texas Children’s Hospital Foundation Chair in Pediatric Cardiovascular Research and by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute. The authors are grateful to Drs Yasuo Ono, Teiji Akagi, Toshiharu Miyake, Hiromichi Hamada, Masaru Terai, Hiroshi Mito, Yasuo Murakami, Takehiko Ishida, Masaki Nii, Yasuhiko Tanaka, Tohru Matsushita, Takeshi Isobe, Hiroshi Sugiyama, Hitoshi Horigome, and Masayuki Matsumoto for patient referrals; Melba Koegele and Kristin Chapman for expert technical assistance; and Dr Partha Sen for the DNA sequence analysis.

Footnotes

Drs Ichida and Tsubata and Drs N.E. Bowles and Towbin contributed equally to this work.